Plant cell wall degrading enzymes are carbohydrate-active enzymes that have been classified in different families based on homology criteria [http://www.cazy.org/, Cantarel et al., 2009, Nucleic Acids Res 37: D233-D238].
Pectate lyases (EC 4.2.2.2), are an important group of plant cell wall degrading enzymes. They cleave pectin using an eliminative cleavage of (1→4)-alpha-D-galacturonan yielding oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups at their non-reducing ends. They are mainly produced by plant pathogens and plant-associated organisms, and only rarely by animals. Pectate lyases are also commonly produced in bacteria, either by bacteria living in close proximity with plants or by gut bacteria that find plant material in the digestive tract of their hosts. [Hugouvieux-Cotte-Pattat et al., Environmental Microbiology reports (2014) doi 10, 1111/1758-2229, 12166].
Pectate lyases favor pectate, the anion, over pectin, the methylated ester, which is the preferred substrate of pectin lyase EC 4.2.2.10. Pectate lyases are also known under different names, such as alpha-1,4-D-endopolygalacturonic acid lyase, endo-alpha-1,4-polygalacturonic acid lyase, endogalacturonate transeliminase, endopectin methyltranseliminase, pectate transeliminase, pectic acid lyase, pectic acid transeliminase, pectic lyase, pectin trans-eliminase, PGA lyase, polygalacturonate lyase, polygalacturonic acid lyase, polygalacturonic acid trans-eliminase, polygalacturonic transeliminase and PPase-N.
When pectate lyases are used in industrial processes, it is often advantageous that they are stable at higher temperatures (thermostable) and resistant to alkaline conditions. Thermostable alkaline pectate lyases for instance have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. Such enzymes have been described, and have been isolated and characterized from bacterial sources, mainly Bacillus [Swarupa Rani Chiliveri et al., Carbohydrate Polymers (2014), 111: 264-272, Zhou et al., Appl Environ Microbiol (2015) 81: 5714-5723].
Cleavage by pectate lyases requires the presence of cations, such as manganese, nickel, iron, cobalt or calcium ions [Celia Marin-Rodriguez et al., J. Exp. Bot. (2002) 53: 2115-2119, Hugouvieux-Cotte-Pattat et al., Environmental Microbiology reports (2014) doi 10, 1111/1758-2229, 12166], with only rare exceptions [Kazemi-Pour et al., Proteomics (2004) 10: 3177-3186].
Recently, a thermostable pectate lyase was isolated from Bacillus, cloned, sequenced and characterized [Takao et al, Biosci. Biotechnol. Biochem. (2000) 64: 2360-2367, Takao et al., Biosci. Biotechnol. Biochem. (2001) 65: 322-329].
This enzyme was described to be thermostable when produced in a homologous expression system in Bacillus subtilis, and capable of resisting pre-incubation for 30 minutes at 70 degrees Celsius [Takao et al., Biosci. Biotechnol. Biochem. (2001) 65: 322-329]. However, pre-incubation at 80 degrees Celsius completely abolished the enzymatic activity. Because many industrial processes are preferably performed at temperatures above 70 degrees Celsius, there is a need in the art for even more thermostable or thermoresistant polypeptides with pectate lyase activity.